P-glycoprotein (P-gp), encoded by the mdr1a gene, is an ATP-dependent plasma membrane protein that is expressed in abundance on the blood-brain barrier (BBB). P-gp limits the CNS influx and retention of a variety of lipophilic compounds. We hypothesized that brain bilirubin content after an i.v. bilirubin infusion would be increased in P-gp-deficient mdr1a null mutant transgenic mice (mdr1a(-/-)) compared with controls. Eighteen mdr1a(-/-) null mutant and 18 P-gp-sufficient wild type mice (+/+) were anesthetized and 50 mg/kg bilirubin infused through the tail vein. Brain bilirubin content (mean ± SEM) 10 min after infusion was significantly higher in mdr1a(-/-) (18.1 ± 2.4 nmol/g) compared with (+/+) mice (10.4 ± 1.0 nmol/g). Brain bilirubin content declined 60 min after infusion but remained higher in mdr1a(-/-) (10.3 ± 1.4 nmol/g) compared with (+/+) mice (5.3 ± 0.9 nmol/g). Brain bilirubin clearance did not differ between groups (t1/2 ∼ 55 min). We conclude that P-gp-deficient mdr1a(-/-) mice have significantly higher brain bilirubin content compared with controls after an i.v. bilirubin load. These data suggest that 1) bilirubin is a substrate for P-gp and 2) the increased brain bilirubin content in mdr1a(-/-) mice is due to enhanced brain bilirubin influx. We speculate that BBB P-gp provides a protective effect against bilirubin neurotoxicity by reducing brain bilirubin influx.